阅读说明：本技术 一种pH/低氧双响应释药协同光动力治疗的纳米复合物及其制备方法 (Nano compound for pH/hypoxia dual-response drug release synergistic photodynamic therapy and preparation method thereof ) 是由 高瑜 李旭东 陈海军 王俊 于 2019-12-05 设计创作，主要内容包括：本发明公开了一种pH/低氧双响应释药协同光动力治疗的纳米复合物及其制备方法。所述纳米材料是通过在壳聚糖表面修饰低氧响应基团2-(2-硝基-1H-咪唑-1-基)乙酸(NA)形成的Cs-NA纳米材料,并与光敏剂相结合,形成纳米复合物。该纳米复合物可在肿瘤酸性和低氧环境下响应释药,并兼具分子靶向作用和光动力疗效,实现光动力治疗和分子靶向治疗的结合,提高光敏剂在肿瘤部位的积聚并降低光毒性。(The invention discloses a pH/hypoxia dual-response drug release and photodynamic therapy nanocomposite and a preparation method thereof. The nano material is a Cs-NA nano material formed by modifying a hypoxia response group 2- (2-nitro-1H-imidazole-1-yl) acetic acid (NA) on the surface of chitosan, and is combined with a photosensitizer to form a nano compound. The nano-composite can respond to drug release in acidic and low-oxygen environments of tumors, has both molecular targeting effect and photodynamic curative effect, realizes the combination of photodynamic treatment and molecular targeting treatment, improves the accumulation of photosensitizer at tumor positions and reduces phototoxicity.)

1. A pH/hypoxia double-response drug release synergistic photodynamic therapy nano-composite is characterized in that: the nano-composite is obtained by taking chitosan Cs-NA modified by nitroimidazole acetate as a carrier and carrying a photosensitizer with negative electricity, wherein the chitosan Cs-NA modified by nitroimidazole acetate is obtained by modifying amino of a chitosan Cs sugar chain with 2- (2-nitroimidazole-1-yl) acetic acid NA, and the grafting rate of 2- (2-nitroimidazole-1-yl) acetic acid is 10% -30%.

2. The nanocomposite of claim 1, wherein: the particle size of the nano-composite is 70-150 nm.

3. The nanocomposite of claim 1, wherein: the molecular weight of the chitosan is 6-1000 kDa.

4. The nanocomposite of claim 1, wherein: the negative photosensitizer comprises one of rose bengal, rose bengal derivatives, indocyanine green, and hematoporphyrin.

5. The nanocomposite as claimed in claim 4, wherein: the rose bengal derivative is a rose bengal derivative with a side chain containing carboxyl, and the carbon number of the side chain is 8.

6. A method of preparing the nanocomposite of claim 1, wherein: the method comprises the following steps:

step (1): dissolving chitosan Cs in 0.1wt% acetic acid solution, sequentially adding NA, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride EDCI and N-hydroxysuccinimide NHS, stirring at room temperature for 12 hours, filling the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain a white flocculent Cs-NA carrier;

step (2): dissolving a Cs-NA carrier in 0.1wt% acetic acid solution, placing the solution into a round-bottom flask, dropwise adding DMSO solution containing a photosensitizer with negative electricity at the dropping speed of 1-2 drops per second, stirring the solution at room temperature in a dark place for reaction for 12 hours, placing the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain the nano-composite.

7. The method of claim 6, wherein: in step 1), Cs: NA: EDCI: the molar ratio of NHS was 1: 1-3: 4: 4.

8. the method of claim 6, wherein: in the nano-composite, the mass ratio of the Cs-NA to the negative photosensitizer is 1: 1-1.5: 1.

9. the method of claim 6, wherein: the cut-off molecular weight of the dialysis bag in the steps (1) and (2) is 8000-14000 Da.

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a chitosan nano material for photodynamic therapy and a preparation method thereof.

Background

Photodynamic therapy (PDT) has been applied as a novel minimally invasive treatment for a variety of cancers. Photosensitizers (PSs) are excited under excitation of specific wavelengths of excitation light, transferring energy to the surrounding oxygen, generating highly toxic Reactive Oxygen Species (ROS), killing tumor cells (Robertson C A, Evans D H, Abrahamse H. Photodynamic therapy (PDT): A short review on cellular mechanisms and cancer applications for PDT [ J ]. Journal of Photochemistry and Photobiology B: Biology, 2009, 96(1): 1-8.). Compared with the traditional operations, chemotherapy and radiotherapy, the photodynamic therapy has the advantages of small wound, higher specificity and small damage to surrounding tissues, and needs specific laser excitation to reduce the toxic and side effects on normal tissues. However, photodynamic therapy has its own drawbacks, and patients receiving photodynamic therapy need to hide in the dark for a long time to avoid phototoxicity, and patient compliance is low.

Hypoxia is a hallmark feature of the tumor microenvironment resulting from aberrant angiogenesis, vascular damage and dysregulation of lymphatic drainage in solid tumors. The oxygen concentration in hypoxic tumor tissue is about (5 mm Hg), which is significantly lower than the hypoxic level in normal tissue (70 mm Hg).

The invention develops a novel chitosan nano material for high-efficiency treatment of lung cancer. Nitroimidazole acetate is coupled on chitosan chains through EDCI/NHS catalyzed amidation reaction to obtain the nano-composite with functions of hypoxia and acidic pH response release and photodynamic therapy.

Disclosure of Invention

The invention aims to provide a preparation method of a controlled-release nano composite medicine with low phototoxicity. The nitroimidazole group hypoxia response and the chitosan acidic pH response are utilized to actively target the tumor site for release, so that the accumulation of the drug on the tumor site is increased, the problem of high phototoxicity of the traditional photosensitizer is solved, and the curative effect of photodynamic therapy is improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

the nano-composite is obtained by taking chitosan Cs-NA modified by nitroimidazole acetate as a carrier and carrying a negative photosensitizer, wherein the chitosan Cs-NA modified by nitroimidazole acetate is obtained by modifying 2- (2-nitroimidazole-1-yl) acetic acid NA on amino of a chitosan Cs sugar chain, and the grafting rate of 2- (2-nitroimidazole-1-yl) acetic acid is 10% -30%.

The particle size of the nano-composite is 70-150 nm.

The molecular weight of the chitosan is 6-1000 kDa.

The negative photosensitizer comprises one of Rose Bengal (RB), Rose Bengal Derivative (RBD), indocyanine green (ICG), and Hematoporphyrin (HP). Wherein the rose bengal RBD is Rose Bengal Derivative (RBD) with side chain containing carboxyl, and the side chain has 8 carbon atoms.

The preparation method of the nano-composite comprises the following steps:

step (1): dissolving chitosan Cs in 0.1wt% acetic acid solution, sequentially adding NA, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDCI and N-hydroxysuccinimide NHS, stirring at room temperature for 12 hours, filling the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain a white flocculent Cs-NA carrier; the reaction steps are as follows:

step (2): dissolving a Cs-NA carrier in 0.1wt% acetic acid solution, placing the solution into a round-bottom flask, dropwise adding DMSO solution containing a photosensitizer with negative electricity at the dropping speed of 1-2 drops per second, stirring the solution at room temperature in a dark place for reaction for 12 hours, placing the reaction solution into a dialysis bag for dialysis, and freeze-drying to obtain the nano-composite.

In the step (1), Cs: NA: EDCI: the molar ratio of NHS was 1: 1-3: 4: 4.

in the nano-composite, the mass ratio of the Cs-NA to the negative photosensitizer is 1: 1-1.5: 1.

the cut-off molecular weight of the dialysis bag in the steps (1) and (2) is 8000-14000 Da.

The nano-composite of the invention is used for the targeted therapy and photodynamic therapy of tumor cells.

The structural formulas of Cs-NA, RBD and RB in the invention are shown in the specification. The RBD can be prepared by a method described in the literature (Sugita N, Kawabata K I, Sasaki K, et al. Synthesis of Amphiphiic derivatives of Rose Bengal and thermal turbine administration [ J ]. bioconjugate ugatechemistry, 2007, 18(3): 866-873.).

First, the principle of the invention: -NH on Chitosan scaffold³⁺Has strong positive charge, can form a stable compound with a photosensitizer with negative charge, achieves the aim of transferring the photosensitizer, realizes the controlled release of the drug, and has better biocompatibility and great clinical application potential.

Secondly, the special pathological environment of tumor cell hypoxia increases the reduction stress, leads to the over-expression of nitroimidazole enzyme, azo reductase and quinone reductase, and utilizes nitroimidazole group to be reduced by nitroreductase in tumor tissue, so as to achieve the purpose of response and release.

Thirdly, the acidic pH value in the tumor tissue is utilized to promote the dissociation of the chitosan structure and promote the drug release.

Fourthly, the photosensitizers RB, RBD, ICG and HP can generate highly toxic reactive oxygen species ROS under the irradiation of exciting light with certain wavelength, and can effectively kill tumor cells.

Fifth, the present invention improves the water solubility of the photosensitizers RB, RBD, ICG, and HP, while reducing its phototoxicity.

The invention has the beneficial effects that:

firstly, the chitosan is used as a delivery carrier, passive targeting is realized by controlling the particle size of the nano-composite, and the medicine is delivered to a tumor part;

secondly, nitroimidazole as a hypoxia response group can be reduced in a hypoxic tumor environment, so that release of the entrapped drug is promoted, and the effects of accumulation of the photosensitizer at a tumor site and reduction of phototoxicity are achieved.

Drawings

FIG. 1 shows the preparation of Cs-NA carrier and raw material Cs in example 2¹H-NMR spectrum;

FIG. 2 is an infrared spectrum of the Cs-NA carrier and the raw Cs prepared in example 2;

FIG. 3 is a particle size peak plot of CBNs and CBDNs prepared in examples 5 and 6;

FIG. 4 is a UV spectrum of CBDNs prepared in example 8;

FIG. 5 is a graph showing the release characteristics of CBDNs of example 9;

FIG. 6 is an in vitro toxicity test of several nanoparticles on PC-9 cells in example 10.

The specific implementation method comprises the following steps:

the present invention is further described below in conjunction with specific examples to assist those of ordinary skill in the art in further understanding the present invention, but are not intended to limit the invention in any way.